Process for quantification of metal amino acid chelates in solutions and solids

ABSTRACT

A process for quantifying the amount of unbound metal and bound metal in solution is provided. A process for quantifying the amount of bound metal amino acid chelate and free ligand in a solid (e.g., dry mixture such as an animal feed) is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 16/408,628filed May 10, 2019 which claims priority to U.S. provisional applicationSer. No. 62/670,202, filed May 11, 2018, the contents of which are eachincorporated herein in its entirety.

BACKGROUND

Metal amino acid chelates and other essential metal/mineral organicsources offer benefits and greater differential bioavailability comparedto inorganic sources in animal and human nutrition. The reaction ofmetals and amino acids to form chelate molecules is a complex reaction.Frequently, manufacturers or commercial synthesizers of these molecules,due to incomplete understanding of chelation chemistry, do not fullyapply any analysis to their synthesis processes which results in acomplex set of non-stable commercial organic type molecules that arecategorized as chelates. The requirement of the heterocyclic ringstructure is not reached, and, instead, a series of complexes areobtained due to uncontrolled reaction between the amino acid and themetal salts or minerals. The result is the formation of other types ofcomplexes that may differ in their chemical structure and therefore intheir bioavailability or diffusion through the small intestine. Asresult of the dose given, the effectiveness may vary according with thesynthesis products obtained in different batches in the absence ofcomplete characterization of the synthesis products. (Ashmead, 2012).

There exists a need for methodologies for the synthesis of metal aminoacid chelates and the establishment of analytical controls that allowmonitoring, characterization, evaluation of comparative stability andcontinuous improvement of the synthesis process to achieve the chemicalstructure desired to ensure an effective, high concentration dose ofbound metal/mineral to an animal. To ensure an effective, highconcentration of bound metal/mineral, the amino acid chelate mustexhibit less reactivity with other antagonistic metal/mineral sources ofthe diet. Due to the unpredictability and lack of stability of aminoacid chelates, production costs remain high. The ability to quantify andasses the stability of amino acid chelates will decrease the cost offormulations by the inclusion of them in a lower quantity and allow forgreater performance of the animal. Additionally, antagonistic reactionsmay be avoided allowing for greater solubility of these compounds at thedifferent physiological conditions (e.g., pH) and avoiding putting atrisk the stability of the premixes due to the inclusion of a highlyreactive species from an unbound metal/mineral source. The has been anexhaustive search for a lower interaction and reactivity of sources ofcalcium, zinc, and copper with other chelating and/or sequesteringsources such as phytic acid present in the cereals of the animal's diet.The metal amino acid chelates are be good alternatives of greaterbioavailability, solubility and less reactivity due to the fact thatthey act as coordination compounds. Thus, the amino acid chelates can beevaluated against a competitor (i.e., exerted by phytic acid) and thestructural stability assessed in solution under simulated physiologicalconditions media.

Phytic acid has the ability to form complexes with essential tracemetals or mineral (Cu, Cr, Zn, Co, Mn, Fe, and Ca), which decreasestheir intestinal absorption and bioavailability in monogastric animalsbecause such animals are not provided with sufficient endogenousphosphatases that are capable of releasing the metals/minerals from thephytate structure. In addition, phytates interact with basic proteinresidues forming complexes, such as protein-phytate andphyto-metal/mineral protein. (NADA M. TAMIM AND ROSELINA ANGEL 2003).

The reaction between phytic acid and an essential trace metal or mineralfavors the formation of highly stable complexes that precipitate inbasic media (e.g., at intestinal pH). As a consequence of precipitation,protein transport located in the intestinal cells for the metallic metalcannot act because the metal is not in the ionic form or soluble form.The same principle is applicable to other substances commonly found inthe diet such as oxalates and phosphates. In fact, calcium, magnesium,zinc, iron and aluminum, react with dietary phosphates to form insolubleprecipitates. The basic environment of the intestine reduces thetendency of the solubility of these when entering into this environment.When the metal/mineral is trapped in a compound of insoluble type, thereis a small probability of a significant absorption of the precipitatedsalt. Thus, there exists a need for analytical testing methods thatvalidate and quantify bound and unbound metal or mineral content underin vitro conditions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a schematic structure of phytic acid in the axial form(Structure I) and in the equatorial form (Structure II). The letters P1, P2, . . . , P6 represent phosphate groups.

FIG. 2 illustrates possible coordinate bonding of metal cations to pairsof oxo di-anions of phytic acid in the axial conformation. Bonding sitesare those that can be constructed from CPK space-filling models.

FIG. 3 is a HPLC chromatogram to 253 nm with peaks 1, 2, 3 copperchelate.

FIG. 4 is copper bisglycinate Masses fragment by UHPLC tandem masses.

FIG. 5 Fragments UHPLC masses/masses zinc triglicinate with a possibleone molecule of water bound and subsequent losses of 44 m/z

FIG. 6 Linearity copper chelate validation developed by HPLC-DAD 253 nm

FIG. 7 XRD pattern of copper amino acid Chelate sample obtained assingle-phase.

FIG. 8 provides an example of Rietveld refinement for copper amino acidchelate sample.

FIG. 9 provides an XRD pattern of zinc amino acid chelate sampleobtained as single-phase.

FIG. 10 provides calibration XRD patterns for copper amino acid chelatesamples.

FIG. 11 provides calibration XRD patterns for zinc amino acid chelatesamples.

FIG. 12 provides calibration curves for copper amino acid chelatesamples.

FIG. 13 provides statistical data ANOVA for rutile compound acting aswitness in the calibration matrix for copper compounds.

FIG. 14 provides calibration curves for zinc amino acid chelate samples.

FIG. 15 provides statistical data ANOVA for rutile compound acting aswitness in the calibration matrix for zinc compounds.

FIG. 16 is a table that shows repeatability data for the evaluation ofcopper bisglycinate HPLC-DAD methodology.

FIG. 17 is a table that shows the accuracy evaluated by triplicate at253 nm by copper bisglycinate at 5 different concentrations is between93 and 106%.

FIG. 18 provides a statistical analysis ANOVA developed to differentlevels of calibration at 253 nm. Experimental F is lower than FCritical.

SUMMARY OF THE DISCLOSURE

According to one aspect, a process for quantifying the amount of unboundmetal and bound metal in solution is provided. The method includes thestep of:

(a) reacting a metal compound with phytic acid in an aqueous solution;

(b) adjusting the pH of the aqueous solution to about 5.0 to about 7.0;

(c) analyzing an aliquot of the solution from step (b) via highperformance liquid chromatography to determine the amount of free ligandand amount of bound metal; and

(d) analyzing total metal content via an inductively coupled plasma(ICP) technique to determine the total amount of metal present insolution and the amount of any insoluble metal precipitate producedafter step (b). According to one embodiment, prior to step (b), theaqueous solution has an initial pH corresponding to the physiological pHof the stomach, gizzard or small intestine. According to one embodiment,prior to step (b), the aqueous solution has an initial pH of about 3.0.According to one embodiment, the bound metal is in the form of a metalamino acid chelate. According to one embodiment, the metal is selectedfrom the group consisting of Cu, Cr, Zn, Co, Mn, Fe, and Ca. Accordingto one embodiment, the pH of the solution in step (b) is adjusted to apH of about 6.0.

According to one aspect, a process for quantifying the amount of boundmetal amino acid chelate and free ligand in a solid is provided. Theincludes the steps of:

(a) qualitatively analyzing a solid sample via x-ray crystallography toobtain an x-ray pattern;

(b) comparing the X-ray pattern of the solid sample from step (a) withone or more x-ray patterns from a reference database; and

(c) determining the amount of bound metal amino acid chelate and freeligand in the solid based on the solid x-ray diffraction intensity.According to one embodiment, the step of comparing the X-ray pattern ofthe solid sample with x-ray patterns from a reference database, can befollowed by a structural refinement process via the Rietveld method.According to one embodiment, the solid is an animal feedstock.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure will now be described more fully hereinafter withreference to exemplary embodiments thereof. These exemplary embodimentsare described so that this disclosure will be thorough and complete, andwill fully convey the scope of the disclosure to those skilled in theart. Indeed, the present disclosure may be embodied in many differentforms and should not be construed as limited to the embodiments setforth herein; rather, these embodiments are provided so that thisdisclosure will satisfy applicable legal requirements. As used in thespecification, and in the appended claims, the singular forms “a”, “an”,“the”, include plural referents unless the context clearly dictatesotherwise.

The term “chelate” as used herein means a molecular entity made up of acentral metal associated with at least one bidentate ligand andoptionally associated with one or more mono- or multi-dentate ligands.In the interaction between the central metal and any of the ligands, thebonds between the ligand and the central metal can include covalentbonds, ionic bonds, and/or coordinate covalent bonds.

The term “ligand” as used herein means a molecular group that isassociated with a central metal atom. The ligand can be any ligandcapable of forming a chelate with a metal. The ligand is preferablyeasily metabolized by an animal.

The term “metal amino acid chelate” as used herein means the productresulting from the reaction of a metal or metal ion from a soluble metalsalt with one or more amino acids. Particularly, a metal amino acidchelate includes the product resulting from the reaction of a metal ionwith amino acids having a mole ratio of one mole metal to one to threemoles of amnio acids to form coordinate covalent bonds. The resultingmolecule may include two or three five-member heterocyclic ringstructures containing a metal ion. The metal ion is attached bycoordinate covalent bonds to two or more nonmetals in the same molecule.The term also refers to a particular class of mineral or mineralcompound. The metal amino acid chelate is nutritionally function. Metalamino acid chelates provide superior bioavailability and lower toxicitycompared to other mineral or mineral compounds. According to oneembodiment, the metal amino acid chelates as provided herein have amolecular weight of less than or equal to 800 daltons (AMU).

The term “metal” refers to any alkaline, alkaline earth, transition, andrare earth, basic, and semi-metals which can coordinate with a ligandand can also be used interchangeably with the term “mineral” whichrefers to a metal compound and includes any trace mineral beneficial toan animal diet (e.g., Copper, Zinc, Cobalt, Manganese, Iron, andCalcium).

The term “phytic acid” used herein refers to the phosphate compoundfound in many plant tissues that is less digested by monogastric animaland by humans. Phytic acid interacts with metal amino acid chelates andmay decrease the absorption of trace metal nutrient bioavailability.

A process for quantifying the metal amino acid chelates in solutions andin solids is provided. According to a particular embodiment, the processprovides for the analysis of chelates and quantifies the bounded metalin solution as well as the stability constant relative to thecompetitor, phytic acid, which is frequently found in animal feeds. Aprocess for quantifying the stability of all metal amino acid chelatesis provided including quantifying the percentage of chelation insolution and in solids (e.g., dry mixture).

The processes as provided herein may be utilized to evaluate commercialchelates and to compare the stability and quality of such commercialchelates. The processes as provided herein also allows for themonitoring of the speed of comparative dilution by modifying the size ofthe crystal in vitro to increase solubility and bioavailability. Theprocesses as provided herein allow for the monitoring and quantificationof the effectiveness of the synthesis (commercial production) of metalamino acid chelates as solids. The processes as provided herein may alsobe utilized for quality control purposes during production of animalfeed. The processes as provided herein utilize HPLC which allowsconfirmation of the stability of a cluster of chelates observed inprevious x-rays and confirms the presence of chelates that remain insolution. The HPLC and X-ray diffraction analysis also allows thedetermining of the crystalline structure information, and therefore,allows an assessment of which crystalline structure prevails in solutionthereby providing an insight as to how to synthesize more bioavailablecompounds.

FIGS. 1 and 2 depicts schematic structures of phytic acid in the axialform (Structure 1) and in the equatorial form (Structure II). Theletters P 1, P2, . . . , P6 represent the phosphate groups which canreact with metal and they can ionized in different order depend on pH.At normal pH range, the phosphate groups of phytic acid are negativelycharged, allowing interaction with positively charged components such asmetals/minerals and proteins. Metal ions may bind with one or morephosphate groups forming complexes of varying solubility. Proteins areable to bind directly with phytic acid through electrostatic charges.Zinc appears to be the most affected by phytic acid because it forms themost stable and insoluble complex. Other metals/minerals and nutrientsthat are affected include calcium (Ca), sodium (Na), iron (Fe),magnesium (Mg), manganese (Mn), and chlorine (CI) and others such as Cu,(copper) and Cr, (chromium)

FIG. 2 shows how metal ion can be kidnapped by phytic acid asconsequence of pH media. Phosphates group are completely ionized atbasic pH and can form macromolecular complex with metal. Research hastraditionally focused on phytic acid's unique structure that providesthe ability to bind metals/minerals, proteins, and starch, and theresulting detrimental effects. Phytic acid has also been attributed tohigh phosphorus excretion by monogastric animals and the resultingenvironmental problems of phosphorus pollution of water and soil.

FIG. 3 depicts a copper chelate chromatogram with three differentpicks—all of them belong to chelate, because in solution copper chelatecan form clusters mono, di, tri gicynates could be present and can formaggregates.

FIG. 4 depicts HPLC masses fragments of copper bisglycinate, thefragment pattern is to molecular pick ion of 211,98 M⁺

FIG. 5 depicts the molecular ion pick 305.8743 (cation formed by theinitial molecule of the analyzed substance less one electron)corresponding to zinc triglicinate with a possible one molecule of boundwater. Subsequent losses of 44 m/z are present due to a loss offragments characterized by a carboxyl group.

FIG. 6 depicts the calibration curve validation of copper bisglycinateobtained by high performance chromatography. A diodes arranged detectorobtained obtained linearity of r² 0.9994 (coefficient correlation). Thecalibration was successfully and illustrated the analytic methodologywas lineal.

FIGS. 7 and 8 depicts copper bisglycinate diffractogram by XRD (Ray Xdiffraction). FIG. 8 depicts the riveted refinement developed based oncrystallographic information from scientific literature and theexperimental copper bisglycinate diffractogram. The final refinement wasmade using the Fullprof software (available from Fullprof suite).

FIG. 9 depicts a zinc bisglycinate diffractogram bases on the rivetedrefinement.

FIGS. 10 and 11 depict a calibration by XRD of copper bisglycinate andzinc bisglycinate. Calibration was built using different samples spikedwith the free ligand, glycine. Rutile was used as internal standard.

FIGS. 12 and 13 depict parameters of validation of the methodologydeveloped by XRD. Linearity of method was adequate to copper and zincbisglycinate. Method repeatability was developed by ANOVA analyses.

According to one embodiment, the process for quantifying the metal aminoacid chelates in solids includes the step of performing X-raydiffraction on a solid. According to a particular embodiment, powderx-ray diffraction analysis is performed. According to one embodiment,the step of performing x-ray diffraction includes the application ofelectromagnetic X-ray wave radiation interacting with matter that ispartially scattered and finally diffracted until fulfillment of theBragg equation. Such methodology may not depend on single-crystal or/andsingle-phase samples to get high quality and accurate results and may,instead, provide a preliminary phase identification via cluster analysisbased on crystallographic reference databases. Such databases arepublicly available. According to a particular embodiment, a crystalstructure determination (e.g., CIF) provides a means to determine thecontent of a crystalline phase by full pattern methods based on theRietveld method. According to one embodiment, Rietveld refinement is aWhole Pattern Fitting Structure Refinement method for structuralanalysis of nearly all classes of crystalline materials not available assingle crystals Such a software approach refines various metricsincluding lattice parameters, peak width and shape, and preferredorientation to derive a calculated diffraction pattern. Once the derivedpattern is nearly identical to an unknown sample data, variousproperties pertaining to that sample can be obtained including, but notlimited to, accurate quantitative information, crystallite size, andsite occupancy factors. The process of refining the pattern iscomputationally intensive, requiring several minutes to calculateresults for a multi-component mixture. According to one embodiment,several polymorphisms may be determined depending on the way of how thesolids are prepared or based on the chemical nature of the organicmetals/minerals themselves.

According to a particular embodiment, the qualitative identification ofphases may be achieved by comparing the X-ray pattern of the solidsample containing metal amino acid chelates with patterns of a referencedatabase (e.g., COD (Crystallography Open Database), ICDD (theinternational Centre for diffraction data) ICSD (Crystal structuredatabase)). After qualitative analysis, semi quantitative phase analysismay be performed. To get reliable results, integral intensities of thereflections may be utilized with a limit of detection at a relativeerror of typically about 1%. According to one embodiment, a standard maybe utilized and a background subtraction from at least a cubicpolynomial as a baseline may be done. The standard may be titaniumoxide, specifically rutile polymorph (95%) from Sigma-Aldrich (99%purity). Mainly reflections of rutile phase do not affect thequantitative determination of the organic metals/minerals analyzed. Thereference intensity ratio (RIR) may be employed as a semi-quantitativeanalysis method as long as RIR values of the structure data isavailable.

According to one embodiment, the selected routine method includes amulti-phase identification, with constituents available as pure phasesand a witness material added because a minimum overlap and similarabsorption may be achieved. From a solid mixture, a calibration curvemay be obtained under identical X-ray diffraction set-up conditions.With this absolute calibration method, a determination of the content ofthe solid chelate sample can be related to the direct measurement ofintensities. These methods by addition of a known amount of the samephase of interest may be controlled by a rutile standard since someevidences of adducts between the metal chelate and the amino acid may befound from a solid state and solution reaction. According to oneembodiment, crystallographic information data may be obtained via theRietveld method.

According to one embodiment, a process of quantifying the metal aminoacid chelates in liquid is provided. According to one embodiment, theprocess includes the step of reacting a sample a liquid sample withphytic acid in an metal such as, for example, Fe, Mn, Cu, Zn, Fe, Co,Cr, or Ca. According to one embodiment, the reaction between the metalsand the phytic acid may be carried out at a pH of typically from about2.0 to about 7.5. According to a particular embodiment, the reactionbetween the metals and the phytic acid may be carried out at a pH oftypically about 3.0.

According to one embodiment, at least one solution of sodium phytate maybe add to one solution of metal/mineral to ensure phytate a ratio ofabout 6:1 of sodium phytate to metal/mineral or excess of phytate. Thecomplex may be completely solubilized at a pH of about 3.0 whichsimulates gastric conditions in monogastric animals and gizzard inpoultry. According to one embodiment, the pH may then be raised totypically about 4.0 to about 7.0. According to a particular embodiment,the pH may then be raised to typically about 6.0. According to oneembodiment, homonuclear complexes of the metal/mineral and phytic acidmay be formed at this pH range. According to one embodiment, suchhomonuclear complexes are released by competition with the metal/mineralwhich is not stabilized by a covalent coordinate bond as a metal aminoacid chelate. An abundant precipitate may then be formed according toone embodiment. According to one embodiment, at a pH of typically about6.0 the source of metal/mineral is not stable and reacts with phyticacid.

According to one embodiment, an aliquot of typically about 5 mL of theremainder solution at a pH of about 6.0 may be passed through an anionicexchange resin. Any unreacted phytic acid may then be eluted withtypically about 10 ml of water. According to one embodiment, the metalamino acid chelate may then be retained and later eluted with 0.6 M HCLand water. According to one embodiment, stable unreacted metal aminoacid chelate may be retained onto the anionic column. Stable unreactedmetal amino acid chelate may then be eluted by a 0.6 M solution of HCLwith about 6 washes of typically about 5 ml. The metal amino acidchelate solution may then be analyzed by high performance liquidchromatograph (HPLC) (diodes arrange or mass detector).

According to an alternative embodiment, a visual testing method may becarried out. According to such an embodiment, phytic acid is reactedwith a metal/mineral source producing a precipitate. The precipitate isthen filtered. Any unstable metal/mineral precipitate may then bequantified by an inductively coupled plasma (ICP) technique. Accordingto one embodiment, the ICP technique is a multi-element analysistechnique that will dissociate a sample into constituent atoms and ionsand exciting them to a higher energy level. The atoms and ion to emitlight at a characteristic wavelength, which can be analyzed viaInductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) orInductively Coupled Plasma Mass Spectrometry (ICP-MS).

According to one embodiment, an eluted sample may be injected into ahigh performance liquid chromatography (HPLC) device. Peaks may thenmonitored for a metal amino acid chelate, metal amino acid chelate andfree ligand. According to one embodiment, the HPLC column is a reversephase column. According to one embodiment, the mobile phase may be anyacceptable mobile phase for the analysis as provided herein includingone or more of ethanol, methanol, acidic water, formic acid, andacetonitrile.

The metal/mineral bounded or stable organic metal/mineral complex andchelate may be quantified using the elute of the retained chelate in thecolumn with mobile phase. Colorless complexes such as Mn, Mg, Zn may beevaluated by passing through an OASIS polymeric cartridge (availablefrom Waters Corporation, Milford, Mass. USA) functionalized bysolid-liquid extraction to remove the ligand. According to oneembodiment, the column allows clusters to be separated due to differentcharges.

Although specific embodiments of the present invention are hereinillustrated and described in detail, the invention is not limitedthereto. The above detailed descriptions are provided as exemplary ofthe present invention and should not be construed as constituting anylimitation of the invention. Modifications will be obvious to thoseskilled in the art, and all modifications that do not depart from thespirit of the invention are intended to be included with the scope ofthe appended claims.

Having generally described the present invention, a furtherunderstanding can be obtained by reference to the examples providedherein for purposes of illustration only and are not intended to belimiting.

EXAMPLE 1—SOLID CHELATE ANALYSIS

The X-ray diffraction (XRD) measurements were obtained using an EmpyreanX′pert XRD diffractometer available from the PANalytical Company. ThisXRD utilized a unique wavelength of Cu Kα1 (1.44556A) and a 3D-solidstate detector with 256 channels under a Bragg-Brentano optical focusinggeometry and a ω-2θ configuration. The step was 0.013 ^(o) and thesource of radiation was excited at 40 mA and 40kV. All samples were rununder the same set-up and a Si standard sample were randomly measured tocheck the system resolution and intensity counting. The samples wereprepared on polymer sample holders of 27 mm of diameter for reflectionmeasurements. The solid surface was completely flat with respect to theXRD ray path to fit the focusing circle of the instrument. The XRD datawere then collected. The identification of the crystalline phases wasachieved by comparing the X-ray pattern of the solid chelate sample withpatterns of a reference database. For Cu and Zn amino acid chelatesamples performed as references as shown in FIG. 7 , a structuralrefinement procedure was carried out in order to get the crystallinestructural information. The input crystallographic information wasobtained from scientific literature and the output file containing thefinal refinement parameters was obtained by using the Fullprof software(available from Fullprof Suite).

After the qualitative analysis was performed, a set of samples wereprepared for a semi quantitative phase analysis. A starting point forthe quantitative analysis depends on the direct proportionality betweenthe intensity of the X-ray diffracted by the metal amino acid chelatephase and the amount in the phase mixture. This first step wasimplemented using calibrations as shown in FIGS. 10 and 11 . Integralintensities of the reflections were utilized along with carefulpreparation and phi-rotation set-up. The limit of detection was statedat a relative error of 1%. Since standards of the metal amino acidchelate was not easily found, a standard sample acting as witness wasselected for this determination. For this example, a titanium oxide wasselected (rutile polymorph (95%) from Sigma-Aldrich (99% purity)).Mainly reflections of rutile phase did not affect the quantitativedetermination of the organic metal/minerals analyzed. The RIR (referenceintensity ratio) could be employed as a semi-quantitative analysismethod as long as RIR values of the structure data is available. Theselected routine method was carried out via a multi-phase identificationwith constituents available as pure phases and a 10% wt. witnessmaterial added because a minimum overlap and similar absorption wereachieved. From the solid mixture, a calibration curve was obtained underidentical XRD set-up conditions as shown in FIG. 10 . The backgroundsubtraction from a fifth grade polynomial as a baseline was carried out.Cubic spline interpolation may be used instead of polynomialinterpolation. Due to this methodology, an absolute calibration methodcould be assumed and a determination of the content of the solid chelatesample can be related to the direct measurement of intensities. Thesemethods by addition of a known amount of the same phase of interest werecontrolled by the rutile standard since some evidences of adductsbetween the metal chelate and the amino acid were found from a solidstate and solution reaction in the literature. Results may be comparedwith the Rietveld method, which is based on theoretical data. Thismethod is based on the minimization of the weighted squares of thedeviations between observed and theoretical intensities of thediffractogram. The statistical data obtained for triplicate are shown inFIGS. 16 and 17 for copper and zinc amino acid chelate samplesrespectively. Based on these results, a high linearity (R2>0.99) showsthe good accuracy of the method. The analysis of variance (ANOVA)analysis for the compound acting as witness, demonstrated the importanceof using this compound as reference for intensity values correctionalong the X-ray diffraction pattern. From above, the semi quantificationof the metal ligates to the amino acid ring—chelate sample—and the freeglycine may be directly measured from the calibration curves for solidsamples. This procedure was able to differentiate the two polymorphs ofglycine, the coordination compounds different to that of chelate sample,the precursors employed from the synthesis reaction and other byproductsthat may be present. The above is a key point to track and control majorstandards for high quality products in the industry. The process asprovided herein is not destructive and samples may be kept for furtheranalysis.

EXAMPLE 2—LIQUID CHELATE ANALYSIS

A liquid sample was reacted with phytic acid. The reaction between thecompounds and the phytic acid was at a pH of 3.0. Phytic acid ionizationbegan via phosphates group in the following order: (a) P1, and P3 at apH of from about 1.5 to about 2.0; (b) P4 and P6 at a pH of from about2.0 to about 2.5; (c) P2 and at a pH of about 2 .5; and (d) P5 at a pHof from about 3.0 to about 5.0 (see FIGS. 1 and 2 ). The pKa's were 1,84, 6, 30, and 9.30 which means that at pH 3.0, the oxygen of thephosphodiester bond were available at this pH and can chelate the metalion. The order of stability of metal-phytate complexes was found to beCu>Zn>Co>Mn>Fe>Ca. Even though Ca has one of the lowest affinities forphytate, Calcium was shown to have the greatest impact because calciumis the metal/mineral present at the highest concentration in the diet.

At a pH of about 3.0, one solution of 4.62 g/L of sodium phytate wasadded to one solution of metal/mineral 0.9% (relation just metal/mineralto complete molecule relation chelate: to ensure Phitate 6:1 or excessof phytate in all cases (Martin C. and Evans W.(1986)). The complex wascompletely solubilized at a pH of 3.0 (simulating gastric conditions inmonogastric animals and gizzard in poultry for example). The pH of thissolution was then changed to a pH of about 6.0. The metal/mineral formedhomonuclear complexes with phytic acid at a pH of 6.0 which wereproduced when the source metal/mineral was released by competition withthe metal/mineral which was not stabilized by a covalent coordinate bondas a metal amino acid chelate thereby obtaining an abundant precipitate.At a pH of about 6.0 (simulated pH intestinal), the source ofmetal/mineral was not stable and reacted with phytic acid.

An aliquot of 5 mL of the remainder solution at a pH of 6.0 was takenand passed through an anionic exchange resin. The unreacted phytic acidwas eluted with 10 ml of water and the metal amino acid chelate wasretained and later eluted with 0.6 M HCL water. Stable unreacted metalamino acid chelate was retained onto the anionic column. The unreactedmetal amino acid chelate was then eluted by a 0.6 M HCL solution with 6washes of 5 mL each. The metal amino acid chelate solution was analyzedby HPLC (diodes arranged or mass detector).

The eluted sample was injected by HPLC with three signals obtained (253or 210 nm range wavelength to detection 200 to 350 nm) at 2.5 min, 3.2min and 3.5 min retention times). All peaks obtained corresponded to ametal amino acid chelate and free ligand (glycine in this example). Thechromatographic setup was based on a HPLC system with a reverse phase C8column×25 cm. The mobile phase included a mix of the following: methanol(5%), acidic water with formic acid (pH 2.72) (80%) and acetonitrile.The time of analyses was 7 minutes. A peak obtained at 2.5 min was acharacteristic signal of a metal amino acid chelate.

We claim:
 1. A process for quantifying the amount of unbound metal andbound metal in solution comprising the step of: (a) reacting a metalcompound with excess phytic acid in an aqueous solution; (b) adjustingthe pH of the aqueous solution to about 5.0 to about 7.0; (c) analyzingan aliquot of the solution from step (b) to determine the amount of freeligand and amount of bound metal; and (d) analyzing total metal contentto determine the total amount of metal present in solution and theamount of any insoluble metal precipitate produced after step (b),wherein prior to step (b), the aqueous solution has an initial pH ofabout 2.0, and wherein the metal is selected from the group consistingof Cu, Cr, Zn, Co, Mn, Fe, and Ca.
 2. The process of claim 1, whereinthe bound metal is in the form of a metal amino acid chelate having amolecular weight of less than or equal to 800 daltons (AMU).
 3. Theprocess of claim 1, wherein the pH of the solution in step (b) isadjusted to a pH of about 6.0.
 4. The process of claim 1, wherein thestep of analyzing an aliquot of the solution to determine the amount offree ligand and amount of bound metal is carried out via highperformance liquid chromatography.
 5. The process of claim 1, whereinthe step of analyzing total metal content to determine the total amountof metal present in solution and the amount of any insoluble metalprecipitate is carried out via an inductively coupled plasma (ICP)technique.
 6. The process of claim 1, wherein the reaction between themetal compound and the excess phytic acid is carried out at a pH ofabout 3.0.
 7. A process for quantifying the amount of bound metal aminoacid chelate and free ligand in a solid comprising the step of:determining the amount of bound metal amino acid chelate and free ligandin the solid based on solid x-ray diffraction intensity of the solidsample, wherein the step of determining amount of bound metal amino acidchelate and free ligand in the solid includes obtaining a calibrationcurve by: performing multi-phase identification with a pure phase and arutile polymorph via x-ray diffraction to obtain a calibration curve;and performing a background subtraction from the calibration curve witha fifth grade polynomial as a baseline.
 8. The process of claim 7,further comprising the steps of: (a) qualitatively analyzing a solidsample via x-ray crystallography to obtain an x-ray pattern; and (b)comparing the X-ray pattern of the solid sample from step (a) with oneor more x-ray patterns from a reference database.
 9. The process ofclaim 8, further comprising the step of structural refinement processvia the Rietveld method.
 10. The process of claim 7, wherein the solidis an animal feedstock.
 11. The process of claim 7, wherein the solidcomprises a dry mixture.
 12. The process of claim 7, wherein the metalis selected from the group consisting of Cu, Cr, Zn, Co, Mn, Fe, and Ca.